We will establish the nature of two novel protein disulfide bond-forming pathways found in certain bacteria and archaea. E. coli and many other bacteria use two enzymes to introduce disulfide bonds into proteins. DsbA directly joins the cysteines of proteins into disulfide bonds while DsbB reoxidizes and thus regenerates active DsbA. Many other bacteria, including Mycobacterium tuberculosis (Mtb), appear to use a homologue of human vitamin K epoxide reductase, VKOR, for disulfide bond formation instead of DsbB. Mtb VKOR can substitute for DsbB in E. coli. We will characterize the mycobacterial VKOR-based disulfide-bond forming system by defining which gene products comprise this oxidative pathway, expressing candidate genes in both E. coli and Mycobacterium smegmatis (which is more tractable than Mtb). In certain archaea, we have obtained evidence for another unusual pathway for disulfide bond formation in the cytoplasm. These archaea contain two VKORs, one cytoplasmically-oriented and apparently involved in cytoplasmic disulfide bond formation. We will verify this pathway and identify the cytoplasmic substrate (a cytoplasmic DsbA?) that this VKOR oxidizes. We will test the proposed role of this VKOR by expressing and manipulating candidate genes both in E. coli and the archaeon Sulfolobus solfataricus. The array of assays and genetic techniques developed in our lab for analyzing disulfide bond formation in the cytoplasm and periplasm will greatly facilitate these studies and those carried out in other laboratories. In our laboratory, we have evolved strains of E. coli that use novel pathways for disulfide bond formation or reduction. We hypothesize that there are other prokaryotes that use these same electron transfer pathways naturally. We will test this hypothesis as follows: we will use bioinformatic analyses that we have developed to assess the capacity of other organisms to make disulfide bonds. We will then use bioinformatic anlaysis of bacterial genomes that have been sequenced to identify prokaryotes that may lack the genes for the pathways E. coli uses for these purposes, but contain genes whose products might constitute one of the novel pathways. We will then study the properties of these candidate genes from other organisms when they are expressed in E. coli and within the native organism itself. We will study DsbB and VKOR function using a chemical biology approach to obtain small molecules that are inhibitors of DsbB and VKOR. Using a highly sensitive assay for disulfide bond formation, we will screen a large library of chemicals for inhibition of E. coli DsbB and Mtb VKOR. These chemicals will be used to dissect out the steps in the action of these two proteins, to define sites of action through resistant-mutations, and to do comparative studies of VKORs and DsbB. The inhibitors will also be screened for their activity as candidates for development as potential antibiotics against tuberculosis and as anti-coagulants. (Human VKOR is a component of the blood coagulation pathway.)